WO2012145746A1 - Compositions and methods for the treatment of neuromyelitis optica - Google Patents
Compositions and methods for the treatment of neuromyelitis optica Download PDFInfo
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- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
Definitions
- the present invention relates generally to the fields of medicine, immunology, neurology and pathology. More particularly, it concerns the development of immunoreagents for use in treating neuromyelitis optica (NMO). 2. Background of the Invention
- AQP4 (aquaporin-4) is a water channel expressed in astrocytes throughout the central nervous system (Lennon et al, 2005) which is involved in water balance in brain (Manley et al., 2000; Papadopoulos et al., 2004) and spinal cord (Saadoun et al., 2008), sensory signal transduction (Li and Verkman, 2001; Lu et al., 2008) and neuroexcitatory phenomena including seizure activity (Binder et al., 2006) and cortical spreading depression (Padmawar et al., 2005) and astrocyte migration and glial scarring (Saadoun et al., 2005; Auguste et al., 2007).
- AQP4 is expressed in astrocytes as two major isoforms: a long (Ml) isoform with translation initiation at Met-1, and a shorter (M23) isoform with translation initiation at Met- 23 (Hasegawa et al, 1994; Jung et al, 1994; Yang et al, 1995; Lu et al, 1996).
- M23 AQP4 assembles in membranes as regular square arrays called orthogonal arrays of particles (OAPs), which were originally seen by freeze-fracture electron microscopy (Landis and Reese, 1974; Wolburg, 1995).
- OAP formation by M23 results from tetramer-tetramer interactions involving residues just downstream of Met-23 at its cytoplasmic N-terminus, while residues in Ml AQP4 just upstream of Met-23 disrupt this interaction (Crane and Verkman, 2009). While Ml does not form OAPs on its own, it can co-assemble with M23 in heterotetramers that limit OAP size (Neely et al, 1999; Furman et al, 2003; Crane et al. (2009); Tajima et al, 2010). The biological significance of OAP formation by AQP4 remains unknown, with speculated functions including cell-cell adhesion, enhanced AQP4 water permeability, and AQP4 polarization to astrocyte end- feet.
- NMO-IgG neuroinflammatory demyelinating disease neuromyelitis optica
- the presence of NMO-IgG is specific for NMO, and in some reports serum NMO-IgG titers correlate with NMO disease activity (Matiello et al, 2008; Jarius et al, 2008). Studies in rodents suggest that NMO-IgG is pathogenic in NMO.
- Human NMO-IgG produces many features of NMO disease in rats with pre-existing experimental autoimmune encephalomyelitis (Bennett et al, 2009; Bradl et al, 2009) or pre-treated with complete Freund's adjuvant (Kinoshita et al, 2010), and in naive mice when injected together with human complement (Saadoun et al, 2010). These animals develop characteristic NMO lesions with neuroinflammation, perivascular deposition of activated complement, demyelination, and loss of astrocyte GFAP and AQP4 immunoreactivity. At present, there remain limited treatments for symptoms of NMO with no known therapies that prevent the underlying inflammatory event.
- a method of treating a subject with neuromyelitis optica (NMO) spectrum disease comprising administering to the subject an reagent comprising an anti-aquaporin-4 (AQP4) antibody or an antigen binding fragment thereof, wherein the reagent lacks effector functions of an intact antibody.
- the subject may be a human subject.
- Administering may comprise intraocular, intra-arterial, subcutaneous, intravenous administration or intrathecal route of administration.
- the reagent may comprise a mutated Fc region lacking effector functions, such as an IgGl sequence that contains L234A/L235A amino acid substitutions, a L234A/L235A/G237A amino acid substitution, and/or a K322A amino acid substitution in the Fc region.
- the reagent may also comprises IgG2 or IgG3 sequences with substitutions in the Fc region that abrogate effector functions such as complement activation.
- the reagent may be an IgG4 antibody.
- the reagent may comprise a chemically modified Fc region.
- the reagent may also comprise an antibody Fab fragment and lacks an Fc region, or an antibody Fab fragment fused to a non-antibody protein segment.
- the reagent may also comprise a single chain antibody or F(ab') 2 .
- the antibody may be optimizied for AQP4 binding by mutating certain residues in the antigen-binding region.
- Treating may comprise reducing one or more of retinal ganglion cell death, optic nerve injury, spinal cord injury, or axonal transection. Treating may comprise reducing one or more of optic nerve demyelination, spinal cord demyelination, astrocyte death or oligodendrocyte death.
- the reagent may be administered more than once, including chronically and daily. The reagent may be administered upon onset of or following an NMO attack, such as within about 1 hour, 6 hours, 12 hours, 24 hours or two days of an NMO attack.
- the method may further comprise administering to the subject a second agent that treats one or more aspect of NMO. The second agent may be administered at the same time as the reagent, or either before or after the reagent.
- the method may further comprise assessing the patient for positive NMO-lgG (AQP4) serology.
- the subject may exhibit positive NMO-lgG (AQP4) serology.
- the subject may exhibit one or more of transverse myelitis, optic neuritis or other unrelated neurologic dysfunction (e.g., protracted nausea or vomiting).
- inventions include a method of chronically treating a subject to prevent or reduce exacerbations of neuromyelitis optica (NMO) spectrum disease in a subject comprising administering to said subject an reagent comprising an anti-aquaporin-4 (AQP4) antibody or an antigen binding fragment thereof, wherein said reagent lacks effector functions of an intact antibody, and a method of preventing or inhibiting the progression of neuromyelitis optica (NMO) spectrum disease in a subject comprising administering to said subject an reagent comprising an anti-aquaporin-4 (AQP4) antibody or an antigen binding fragment thereof, wherein said reagent lacks effector functions of an intact antibody.
- AQP4 anti-aquaporin-4
- a reagent comprising an anti-aquaporin-4
- the reagent may comprise a mutated Fc region lacking effector functions, such as one comprising an IgGl sequence that contains L234A/L235A amino acid substitutions, a L234A/L235A/G237A amino acid substitution, and/or comprising an IgGl sequence having a K322A substitution, or even a mutated IgG2 or IgG3 Fc region.
- The may be reagent an IgG4 antibody.
- the reagent may comprise a chemically-modified Fc region.
- the reagent may comprise an antibody Fab fragment and lacks an Fc region.
- the reagent may comprise an antibody Fab fragment fused to a non-antibody protein segment.
- the reagent may comprise a single chain antibody or F(ab) 2 . It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein.
- the antibody or antigen binding fragment of the antibodies set forth above may comprise a light chain variable sequence according to SEQ ID NO: 6 or a sequence having 95%, 90%), 85%), or 80%> identity to SEQ ID NO:6, and a heavy chain sequence according to SEQ ID NO:8, or a sequence having 95%, 90%, 85%, or 80% identity to SEQ ID NO:8; or a light chain variable sequence according to SEQ ID NO: 10 or a sequence having 95%, 90%>, 85%), or 80%) identity to SEQ ID NO: 10, and a heavy chain sequence according to SEQ ID NO: 12 or a sequence having 95%, 90%, 85%, or 80% identity to SEQ ID NO:12; or a light chain variable sequence according to SEQ ID NO: 19 or a sequence having 95%, 90%>, 85%, or 80% identity to SEQ ID NO: 19, and a heavy chain variable sequences SEQ ID NO: 17 or a sequence having 95%, 90%, 85%, or 80% identity to SEQ ID NO: 17.
- the antibody antigen binding fragment may be encoded by a light chain variable sequence according to SEQ ID NO:5 or a sequence having 85%, 80%, 75% or 70% identity to SEQ ID NO:5, and a heavy chain sequence according to SEQ ID NO:7, or a sequence having 85%, 80%, 75% or 70% identity to SEQ ID NO:7; or encoded by a light chain variable sequence according to SEQ ID NO:9 or a sequence having 85%, 80%, 75% or 70% identity to SEQ ID NO:9, and a heavy chain sequence according to SEQ ID NO: 11 or a sequence having 85%, 80%, 75% or 70% identity to SEQ ID NO: 11; or encoded by a light chain variable sequence according to SEQ ID NO:20 or a sequence having 85%, 80%, 75% or 70% identity to SEQ ID NO:20, and a heavy chain sequence according to SEQ ID NO: 18 or a sequence having 85%, 80%, 75% or 70% identity to SEQ ID NO: 18.
- the antibody antigen binding fragment
- FIGS. 1A-B Schematic of the two-color ratio imaging method for quantitative measurement of NMO-IgG binding to AQP4 iso forms.
- FIG. 1A AQP4 monomers (cylinders) shown as assembling into tetramers (top) or OAPs (bottom).
- NMO-IgG green binds AQP4 at an extracellular domain, and a reference AQP4 antibody (red) binds on the cytoplasmic side.
- the reference AQP4 antibody binds to the C-terminus of AQP4, independent of the AQP4 N-terminal isoform and OAP formation.
- FIGS. 2A-D Characterization of stably transfected, AQP4-expressing
- FIG. 2A Confocal fluorescence images show U87MG cells stably expressing Ml (top) or M23 (bottom) and labeled with NMO-IgG (green) and C- terminal anti-AQP4 antibody (red).
- FIG. 2B Total internal reflection fluorescence images show distinct OAPs in M23 -expressing cells (bottom), and a smooth fluorescence staining pattern in Ml-expressing cells (top).
- FIG. 2C AQP4 immunoblot following blue-native gel electrophoresis (top) and Tricine SDS-PAGE (bottom) of stable AQP4-expressing U87MG cell lysates.
- G/R green- to-red fluorescence ratios
- FIGS. 3A-B Differential binding of NMO-IgG in NMO patient serum to Ml vs. M23 AQP4.
- FIG. 3A Ml and M23 expressing U87MG cells stained with 5% NMO serum (green) from four patients, and with reference AQP4 antibody (red).
- FIGS. 4A-B Differential binding of purified monoclonal NMO-IgGs to Ml vs. M23 AQP4.
- FIG. 4A Representative fluorescence micrographs for binding of rAb-53 and rAb-58 (green) as a function of concentration, together with reference AQP4 antibody (red).
- FIGS. 5A-C Binding of NMO-IgG to mixtures of Ml and M23 AQP4. and to M23 mutants containing OAP-disrupting mutations.
- FIG. 6A-D Mechanism of increased NMO-IgG binding affinity to array- assembled AQP4.
- FIG. 6A Human IgG and AQP4 crystal structures (Harris et ah, 1998; Ho et al., 2009) showing relative size of the AQP4 tetramer compared with spacing between Fab binding sites in whole IgG.
- FIG. 6B Predictions of bivalent vs. monovalent binding mechanisms.
- AQP4 monomers (cylinders) are shown as assembled in tetramers (Ml) or OAPs (M23).
- NMO-IgG green
- FIG. 6A Human IgG and AQP4 crystal structures (Harris et ah, 1998; Ho et al., 2009) showing relative size of the AQP4 tetramer compared with spacing between Fab binding sites in whole IgG.
- FIG. 6C Binding of monoclonal mouse anti-Myc to cells expressing Myc-tagged Ml vs. M23 AQP4.
- FIGS. 7A-C High-affinity monoclonal, recombinant anti-AQP4 antibody for aquaporumab therapy.
- FIG. 7A Crystal structure of AQP4 tetramer shown on the same scale with that of an IgGl antibody.
- FIG. 7B Surface plasmon resonance measurement of recombinant antibody binding to AQP4-reconstituted proteoliposomes showing binding / unbinding kinetics of rAb-53 (left) at different concentrations, and different NMO rAbs (right) at fixed concentration.
- FIG. 7C Binding and unbinding kinetics rAb-53 (25 ⁇ g/ml) to AQP4-expressing U87MG cells.
- FIGS. 8A-D Mutated, non-pathogenic rAb-53 (aquaporumab) blocks binding of pathogenic NMO-IgG to AQP4. (FIG.
- FIG. 8A Schematic of rAb-53 showing heavy (VH) and light (VL) chain variable regions, light chain constant region (CL), and IgGl heavy chain constant regions (CH1-CH3). Locations of amino acid mutations introduced in the CH2 domain to reduce CDC (K322A), ADCC (K326W/E333S) or both (L234A/L235A).
- FIG. 8B Surface plasmon resonance measurements of binding and washout of a mutated rAb-53 (L234A/L235A) to AQP4-reconstituted proteoliposomes.
- FIG. 8D Unrelated monoclonal NMO antibodies and human NMO serum blocks AQP4 binding of Cy3 -labeled rAb-53. Cy3 fluorescence imaged in cells incubated with 20 ⁇ g/ml Cy3-rAb-53 for 1 h in the absence or presence of 10% control (non-NMO) or NMO patient serum, or 100 ⁇ g/ml recombinant NMO monoclonal antibody rAb- 186.
- FIGS. 9A-D Mutated non-pathogenic rAb-53 aquaporumabs prevents CDC and ADCC in NMO-IgG-exposed AQP4-expressing cells.
- FIGS. 9A-D Mutated non-pathogenic rAb-53 aquaporumabs prevents CDC and ADCC in NMO-IgG-exposed AQP4-expressing cells.
- FIG. 9B Assay as in A done with complement + rAb-53, in the presence of 12.5 ⁇ g/ml of the indicated aquaporumabs.
- FIG. 9C Live/dead cell assay after 60 min exposure to control (non-NMO) serum or NMO patient sera in the presence of complement, and the absence or presence of indicated aquaporumabs.
- FIG. 9D ADCC assay done using AQP4-expressing CHO cells incubated with NK-cells together with control (non-NMO) mAb or rAb-53 or aquaporumab (individually), or rAb-53 and aquaporumab together.
- FIGS. lOA-C Aquaporumab reduces NMO-like lesions in mouse brain in vivo produced by intracerebral injection of NMO-IgG and human complement. (FIG.
- FIG. 10B AQP4 and myelin loss quantified as % area outlined with pink/area outlined with black (S.E.M., 5 mice per group, * P ⁇ 0.01).
- FIG. IOC % myelin and AQP4 loss shown for five pairs of mice, each pair injected with NMO-IgG from a different NMO patient with human complement, without or with aquaporumab .
- FIGS. 11A-B Aquaporumab reduces NMO-like lesions produced by NMO- IgG and human complement in ex vivo spinal cord slice cultures.
- FIG. 1 1 A E vivo spinal cord slice culture model in which slices were cultured for 7 days, followed by 3 days in the presence of NMO-IgG (purified IgG from NMO serum) and human complement, without or with aquaporumab (Aqmab). Immunostaining shown for
- Controls include non-NMO IgG, NMO-IgG or Aqmab alone, Aqmab with complement, and slice cultures from AQP4 null mice.
- NMO patient serum and a recombinant monoclonal NMO-IgG were each able to bind to both the M23 and Ml isoforms of AQP4 (Crane et al, 2009), which contradicted an earlier study reporting undetectable binding to Ml AQP4 of serum from one NMO patient (Nicchia et al., 2009).
- a prior report that analyzed a single NMO serum specimen concluded that OAPs are the sole target of NMO-IgG (Nicchia et al, 2009).
- the inventors used quantitative ratio imaging to measure NMO autoantibody binding to AQP4 in which NMO-IgG binding, as revealed by a fluorescent secondary antibody, was normalized to total AQP4 protein using an antibody directed against the AQP4 C-terminus. For these studies, the inventors identified a human astrocyte-derived cell line that expressed AQP4 in a plasma membrane pattern after transfection.
- the strategy to assess independently the AQP4 isoform and OAP specificities of NMO-IgG binding was to express Ml and M23 AQP4 in different ratios, or an M23 mutant containing an OAP-disrupting, single amino acid substitution in its N-terminus.
- Aquaporumab comprises a tight-binding anti-AQP4 Fab and a mutated Fc that lacks functionality for complement- and cell-mediated cytotoxicity.
- aquaporumab blocked binding of NMO-IgG in human sera, reducing to near zero complement- and cell-mediated cytotoxicity.
- Aquaporumab prevented the development of NMO-like lesions in an ex vivo spinal cord slice model of NMO and in an in vivo mouse model of NMO produced by intracerebral injection of NMO-IgG and complement. Aquaporumab alone did not cause pathology. The broad efficacy of aquaporumab inhibition is likely due to steric competition because of its large physical size compared to the extracellular domain of AQP4. These results provide support for aquaporumab therapy of NMO.
- NMO Neuromyelitis optica
- Neuromyelitis optica also known as Devic's disease or Devic's syndrome
- Devic's disease is an autoimmune, inflammatory disorder in which a person's own immune system attacks the optic nerves and spinal cord. This produces an inflammation of the optic nerve (optic neuritis) and the spinal cord (myelitis). Although inflammation may also affect the brain, the lesions are different from those observed in the related condition multiple sclerosis (MS). Spinal cord lesions lead to varying degrees of weakness or paralysis in the legs or arms, loss of sensation (including blindness), and/or bladder and bowel dysfunction.
- MS multiple sclerosis
- NMO is a rare disorder which resembles MS in several ways, but requires a different course of treatment for optimal results. NMO has also been suggested to be a variant form of acute disseminated encephalomyelitis. The target of the autoimmune attack in at least some patients with NMO has been identified - it is a protein of the nervous system cells called aquaporin 4 or AQP4.
- NMO neurodegenerative disease
- the visual impairment usually manifests as decreased visual acuity, although visual field defects, or loss of color vision may occur in isolation or prior to formal loss of acuity.
- Spinal cord dysfunction can lead to muscle weakness, reduced sensation, or loss of bladder and bowel control.
- the typical patient has an acute and severe spastic weakness of the legs (paraparesis) or all four limbs (tetraparesis) with sensory signs, often accompanied by loss of bladder control.
- NMO is similar to MS in that there is immune-mediated destruction of the myelin surrounding nerve cells. Unlike standard MS, the attacks are not targeted against the myelin producing cells (oligodendrocytes) or primarily mediated by the immune system's T cells but rather by antibodies called NMO-IgG, or simply NMO antibodies. These antibodies target AQP4 in the cell membranes of astrocytes which acts as a channel for the transport of water across the cell membrane. AQP4 is found in the processes of the astrocytes that surround the blood-brain barrier, a system responsible for preventing substances in the blood from crossing into the brain. The blood-brain barrier is weakened in NMO, but it is currently unknown how the NMO-IgG immune response leads to oligodendrocyte death and demyelination.
- NMO neurodegenerative disease 2019
- the damage in the spinal cord can range from inflammatory demyelination to necrotic damage of the white and grey matter.
- the inflammatory lesions in NMO have been classified as type II lesions (complement mediated demyelination), but they differ from MS pattern II lesions in their prominent perivascular distribution. Therefore, the pattern of inflammation is often quite distinct from that seen in MS.
- the Mayo Clinic proposed a revised set of criteria for diagnosis of NMO in 2006. The new guidelines for diagnosis require two absolute criteria plus at least two of three supportive criteria being:
- NMO-IgG seropositive status The NMO-IgG test checks the existence of antibodies against the aquaporin 4 antigen.
- the NMO spectrum is now believed to consist of:
- Optic neuritis or myelitis associated with lesions in specific brain areas such as the hypothalamus, periventricular nucleus, and brainstem
- NMO neuro-inflammatory disorder
- MS infrequently presents as transverse myelitis
- oligoclonal bands in the CSF, as well as white matter lesions on brain MRI are uncommon in Devic's disease but occur in over 90% of MS patients.
- antiviral immune response distinguishes multiple sclerosis and neuromyelitis optica.
- NMO has been associated with many systemic diseases, based on anecdoctal evidence of some NMO patients with a comorbid condition. Such conditions include: collagen vascular diseases, autoantibody syndromes, infections with varicella-zoster virus, Epstein-Barr virus, and HIV, and exposure to clioquinol and antituberculosis drugs.
- the disease can be monophasic, i.e., a single episode with permanent remission.
- NMO National Metalomyolism
- Asian optic-spinal MS which constitutes 30%> of the cases of MS in Japan
- NMO differences between optic-spinal and classic MS in Japanese patients.
- MS is rare, but when it appears it often takes the form of optic-spinal MS.
- the majority of NMO patients have no affected relatives, and it is generally regarded as a non-familial condition.
- monoclonal antibodies binding to AQP4 will have utilities in several applications. These include the production of diagnostic kits for use in detecting and diagnosing NMO, as well as for treating NMO. In these contexts, one may link such antibodies to diagnostic or therapeutic agents, use them as capture agents or competitors in competitive assays, or use them individually without additional agents being attached thereto. The antibodies may be mutated or modified, as discussed further below. Methods for preparing and characterizing antibodies are well known in the art (see, e.g., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; U.S. Patent 4,196,265).
- the methods for generating monoclonal antibodies generally begin along the same lines as those for preparing polyclonal antibodies.
- the first step for both these methods is immunization of an appropriate host or identification of subjects who are immune due to prior natural infection.
- a given composition for immunization may vary in its immunogenicity. It is often necessary therefore to boost the host immune system, as may be achieved by coupling a peptide or polypeptide immunogen to a carrier.
- exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA).
- KLH keyhole limpet hemocyanin
- BSA bovine serum albumin
- Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as carriers.
- Means for conjugating a polypeptide to a carrier protein are well known in the art and include glutaraldehyde, m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimyde and bis-biazotized benzidine.
- the immunogenicity of a particular immunogen composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants.
- adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvants and aluminum hydroxide adjuvant.
- the amount of immunogen composition used in the production of polyclonal antibodies varies upon the nature of the immunogen as well as the animal used for immunization.
- a variety of routes can be used to administer the immunogen (subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal).
- the production of polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization. A second, booster injection, also may be given. The process of boosting and titering is repeated until a suitable titer is achieved.
- the immunized animal can be bled and the serum isolated and stored, and/or the animal can be used to generate MAbs.
- the recombinant antibodies of the present invention are producing using single plasmablasts or b cells isolated from the CSF of affected individuals. Blood can also be used, although plasmablasts are somewhat less prevalent in that fluid.
- the antibody heavy and light chain sequences are identified by RT-PCR. The identified pair of heavy and light chain are then reengineered using standard cloning techniques into expression vectors and transfected into mammalian cell lines to produce antibody.
- MAbs produced by either means may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as FPLC or affinity chromatography.
- Fragments of the monoclonal antibodies of the invention can be obtained from the purified monoclonal antibodies by methods which include digestion with enzymes, such as pepsin or papain, and/or by cleavage of disulfide bonds by chemical reduction.
- monoclonal antibody fragments encompassed by the present invention can be synthesized using an automated peptide synthesizer.
- RNA can be isolated from the hybridoma line and the antibody genes obtained by RT-PCR and cloned into an immunoglobulin expression vector.
- combinatorial immunoglobulin phagemid libraries are prepared from RNA isolated from the cell lines and phagemids expressing appropriate antibodies are selected by panning using viral antigens.
- Antibodies according to the present invention may be defined, in the first instance, by their binding specificity, which in this case is for AQP4. Those of skill in the art, by assessing the binding specificity/affinity of a given antibody using techniques well known to those of skill in the art, can determine whether such antibodies fall within the scope of the instant claims.
- Such antibodies may be produced by mutating the Fc region of antibodies that exhibit such functions (e.g., IgGl or IgG2 or IgG3), by using antibodies that naturally lack such functions (IgG4), or by chemically modifying any of such antibodies so as to render them ineffective at complement activation and immune cell recruitment.
- the antibodies may be defined by virtue of the region of the structures to which they bind.
- the extracelllar surface and orthogonal arrays of AQP4 provide a unique platform for antibody binding.
- the antibodies may be defined by their variable sequence that determine their binding specificity. Examples are provided below: rAb53 Heavy Chain Sequence (Variable Region) Nucleic Acid (SEQ ID NO: 7): CAGGTGCAGCTGCAGGAGTCGGGCGCAGGACTGGTGAAGCCTTCGGAGACCCTG TCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGTGGTCACTACTGGAACTGGA TCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGGTACATCCATTACAGTG GGAGCACCAACTACAACCCCTCCCTCAAGAGTCGAGTCACCATATCAGTGGACA CGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCTGCGGACACGGC CGTGTATTACTGTGCGAGAGCAGAGGGGAGAGGATGGAGTGCTTTCT ACT ACTA CTACATGGAAGTCTGGGGCAAAGGGTCCACGGTCTCCGTCCTCA rAb53 Heavy Chain Sequence (Variable Region) Protein (SEQ ID NO: 7)
- rAb-53 was originally an IgG2 molecule, and the cloning steps used to construct the mutant IgGl Fc regions retained some portion of the 5 ' IgG2 Fc sequence.
- the sequences are distinct from IgGl at 4 amino acids in the first 35 aa.
- the inventors added a FLAG tag (LEDYKDDDDK; SEQ ID NO: 16). Therefore, the sequence is not technically human IgGl; it is different by 14/340 residues, or 4%.
- the final product is a mutated human IgGl .
- Mutant Human IgGl Fc K322 mutation Nucleic Acid SEQ ID NO:3
- nucleic acid sequences may vary from those set out above in that (a) the variable regions may be segregated away from the constant domains of the light chains, (b) the nucleic acids may vary from those set out above while not affecting the residues encoded thereby, (c) the nucleic acids may vary from those set out above by a given percentage, e.g., 70%, 75%, 80%>, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology, (d) the nucleic acids may vary from those set out above by virtue of the ability to hybridize under high stringency conditions, e.g., 65 °C, 50%> formamide, 0.1 x SSC, 0.1%> SDS, (e) the amino acids may vary from those set out above by a given percentage, e.g., 80%, 85%, 90%,
- reasons such as improved expression, improved cross-reactivity, diminished off-target binding or abrogation of one or more natural effector functions, such as activation of complement or recruitment of immune cells ⁇ e.g., T cells, monocytes or NK cells.
- Hybridomas may cultured, then cells lysed, and total RNA extracted. Random hexamers may be used with RT to generate cDNA copies of RNA, and then PCR performed using a multiplex mixture of PCR primers expected to amplify all human variable gene sequences. PCR product can be cloned into pGEM-T Easy vector, then sequenced by automated DNA sequencing using standard vector primers. Assay of binding and neutralization may be performed using antibodies collected from hybridoma supernatants and purified by FPLC, using Protein G columns.
- Recombinant full length IgG antibodies may be generated by subcloning heavy and light chain Fv DNAs from the cloning vector into a Lonza pConlgGl or pConK2 plasmid vector, transfected into 293 Freestyle cells or Lonza CHO cells, and antibodies were collected an purified from the CHO cell supernatant. Other methods are described in Bennett et al. (2009).
- Lonza has developed a generic method using pooled transfectants grown in CDACF medium, for the rapid production of small quantities (up to 50 g) of antibodies in CHO cells. Although slightly slower than a true transient system, the advantages include a higher product concentration and use of the same host and process as the production cell line.
- pCon VectorsTM are an easy way to re-express whole antibodies.
- the constant region vectors are a set of vectors offering a range of immunoglobulin constant region vectors cloned into the pEE vectors. These vectors offer easy construction of full length antibodies with human constant regions and the convenience of the GS SystemTM.
- Antibody molecules will comprise fragments (such as F(ab'), F(ab') 2 ) that are produced, for example, by the proteolytic cleavage of the mAbs, or single-chain immunoglobulins producible, for example, via recombinant means. Such antibody derivatives are monovalent. In one embodiment, such fragments can be combined with one another, or with other antibody fragments or receptor ligands to form "chimeric" binding molecules. Significantly, such chimeric molecules may contain substituents capable of binding to different epitopes of the same molecule.
- Humanized antibodies produced in non-human hosts in order to attenuate any immune reaction when used in human therapy.
- Such humanized antibodies may be studied in an in vitro or an in vivo context.
- Humanized antibodies may be produced, for example by replacing an immunogenic portion of an antibody with a corresponding, but non- immunogenic portion (i.e., chimeric antibodies).
- Humanized chimeric antibodies are provided by Morrison (1985); also incorporated herein by reference. “Humanized” antibodies can alternatively be produced by CDR or CEA substitution. Jones et al. (1986); Verhoeyen et al. (1988); Beidler et al. (1988); all of which are incorporated herein by reference.
- the antibody is a derivative of the disclosed antibodies, e.g. , an antibody comprising the CDR sequences identical to those in the disclosed antibodies (e.g., a chimeric, humanized or CDR-grafted antibody).
- the antibody is a fully human recombinant antibody.
- the hydropathic index of amino acids may be considered.
- the importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.
- Patent 4,554,101 the following hydrophilicity values have been assigned to amino acid residues: basic amino acids: arginine (+3.0), lysine (+3.0), and histidine (-0.5); acidic amino acids: aspartate (+3.0 ⁇ 1), glutamate (+3.0 ⁇ 1), asparagine (+0.2), and glutamine (+0.2); hydrophilic, nonionic amino acids: serine (+0.3), asparagine (+0.2), glutamine (+0.2), and threonine (-0.4), sulfur containing amino acids: cysteine (-1.0) and methionine (-1.3); hydrophobic, nonaromatic amino acids: valine (-1.5), leucine (-1.8), isoleucine (-1.8), proline (-0.5 ⁇ 1), alanine (-0.5), and glycine (0); hydrophobic, aromatic amino acids: tryptophan (-3.4), phenylalanine (-2.5), and tyrosine (-2.3).
- an amino acid can be substituted for another having a similar hydrophilicity and produce a biologically or immunologically modified protein.
- substitution of amino acids whose hydrophilicity values are within ⁇ 2 is preferred, those that are within ⁇ 1 are particularly preferred, and those within ⁇ 0.5 are even more particularly preferred.
- amino acid substitutions generally are based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
- Exemplary substitutions that take into consideration the various foregoing characteristics are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
- the present invention also contemplates isotype modification.
- isotype modification By modifying the Fc region to have a different isotype, different functionalities can be achieved. For example, changing to IgG 4 can reduce immune effector functions associated with other isotypes.
- Modified antibodies may be made by any technique known to those of skill in the art, including expression through standard molecular biological techniques, or the chemical synthesis of polypeptides. Methods for recombinant expression are addressed elsewhere in this document.
- a Single Chain Variable Fragment is a fusion of the variable regions of the heavy and light chains of immunoglobulins, linked together with a short (usually serine, glycine) linker.
- This chimeric molecule retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of a linker peptide. This modification usually leaves the specificity unaltered.
- These molecules were created historically to facilitate phage display where it is highly convenient to express the antigen binding domain as a single peptide.
- scFv can be created directly from subcloned heavy and light chains derived from a hybridoma.
- Single chain variable fragments lack the constant Fc region found in complete antibody molecules, and thus, the common binding sites ⁇ e.g., protein A/G) used to purify antibodies. These fragments can often be purified/immobilized using Protein L since Protein L interacts with the variable region of kappa light chains.
- Flexible linkers generally are comprised of helix- and turn-promoting amino acid residues such as alaine, serine and glycine. However, other residues can function as well.
- Tang et al. (1996) used phage display as a means of rapidly selecting tailored linkers for single-chain antibodies (scFvs) from protein linker libraries.
- a random linker library was constructed in which the genes for the heavy and light chain variable domains were linked by a segment encoding an 18-amino acid polypeptide of variable composition.
- the scFv repertoire (approx. 5 x 10 6 different members) was displayed on filamentous phage and subjected to affinity selection with hapten. The population of selected variants exhibited significant increases in binding activity but retained considerable sequence diversity.
- the recombinant antibodies of the present invention may also involve sequences or moieties that permit dimerization or multimerization of the receptors.
- sequences include those derived from IgA, which permit formation of multimers in conjunction with the J- chain.
- Another multimerization domain is the Gal4 dimerization domain.
- the chains may be modified with agents such as biotin/avidin, which permit the combination of two antibodies.
- a single-chain antibody can be created by joining receptor light and heavy chains using a non-peptide linker or chemical unit.
- the light and heavy chains will be produced in distinct cells, purified, and subsequently linked together in an appropriate fashion (i.e., the N-terminus of the heavy chain being attached to the C- terminus of the light chain via an appropriate chemical bridge).
- Cross-linking reagents are used to form molecular bridges that tie functional groups of two different molecules, e.g., a stablizing and coagulating agent.
- a stablizing and coagulating agent e.g., a stablizing and coagulating agent.
- dimers or multimers of the same analog or heteromeric complexes comprised of different analogs can be created.
- hetero- bifunctional cross-linkers can be used that eliminate unwanted homopolymer formation.
- An exemplary hetero-bifunctional cross-linker contains two reactive groups: one reacting with primary amine group (e.g., N-hydroxy succinimide) and the other reacting with a thiol group (e.g., pyridyl disulfide, maleimides, halogens, etc.).
- primary amine group e.g., N-hydroxy succinimide
- a thiol group e.g., pyridyl disulfide, maleimides, halogens, etc.
- the cross-linker may react with the lysine residue(s) of one protein (e.g., the selected antibody or fragment) and through the thiol reactive group, the cross-linker, already tied up to the first protein, reacts with the cysteine residue (free sulfhydryl group) of the other protein (e.g., the selective agent).
- cross-linker having reasonable stability in blood will be employed.
- Numerous types of disulfide-bond containing linkers are known that can be successfully employed to conjugate targeting and therapeutic/preventative agents. Linkers that contain a disulfide bond that is sterically hindered may prove to give greater stability in vivo, preventing release of the targeting peptide prior to reaching the site of action. These linkers are thus one group of linking agents.
- SMPT cross-linking reagent
- Another cross-linking reagent is SMPT, which is a bifunctional cross-linker containing a disulfide bond that is "sterically hindered" by an adjacent benzene ring and methyl groups. It is believed that steric hindrance of the disulfide bond serves a function of protecting the bond from attack by thiolate anions such as glutathione which can be present in tissues and blood, and thereby help in preventing decoupling of the conjugate prior to the delivery of the attached agent to the target site.
- thiolate anions such as glutathione which can be present in tissues and blood
- the SMPT cross-linking reagent lends the ability to cross-link functional groups such as the SH of cysteine or primary amines ⁇ e.g., the epsilon amino group of lysine).
- Another possible type of cross-linker includes the hetero-bifunctional photoreactive phenylazides containing a cleavable disulfide bond such as sulfosuccinimidyl-2-(p-azido salicylamido) ethyl-l,3'-dithiopropionate.
- the N-hydroxy- succinimidyl group reacts with primary amino groups and the phenylazide (upon photolysis) reacts non-selectively with any amino acid residue.
- non-hindered linkers also can be employed in accordance herewith.
- Other useful cross-linkers include SATA, SPDP and 2-iminothiolane (Wawrzynczak & Thorpe, 1987). The use of such cross-linkers is well understood in the art. Another embodiment involves the use of flexible linkers.
- U.S. Patent 4,680,3308 describes bifunctional linkers useful for producing conjugates of ligands with amine-containing polymers and/or proteins, especially for forming antibody conjugates with chelators, drugs, enzymes, detectable labels and the like.
- U.S. Patents 5,141,648 and 5,563,250 disclose cleavable conjugates containing a labile bond that is cleavable under a variety of mild conditions. This linker is particularly useful in that the agent of interest may be bonded directly to the linker, with cleavage resulting in release of the active agent. Particular uses include adding a free amino or free sulfhydryl group to a protein, such as an antibody, or a drug.
- U.S. Patent 5,856,456 provides peptide linkers for use in connecting polypeptide constituents to make fusion proteins, e.g., single chain antibodies.
- the linker is up to about 50 amino acids in length, contains at least one occurrence of a charged amino acid (preferably arginine or lysine) followed by a proline, and is characterized by greater stability and reduced aggregation.
- U.S. Patent 5,880,270 discloses aminooxy-containing linkers useful in a variety of immunodiagnostic and separative techniques.
- the antibodies of the present invention may be purified.
- purified is intended to refer to a composition, isolatable from other components, wherein the protein is purified to any degree relative to its naturally-obtainable state.
- a purified protein therefore also refers to a protein, free from the environment in which it may naturally occur.
- substantially purified this designation will refer to a composition in which the protein or peptide forms the major component of the composition, such as constituting about 50%, about 60%, about 70%>, about 80%>, about 90%>, about 95% or more of the proteins in the composition.
- Protein purification techniques are well known to those of skill in the art. These techniques involve, at one level, the crude fractionation of the cellular milieu to polypeptide and non-polypeptide fractions. Having separated the polypeptide from other proteins, the polypeptide of interest may be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suited to the preparation of a pure peptide are ion-exchange chromatography, exclusion chromatography; polyacrylamide gel electrophoresis; isoelectric focusing.
- protein purification include, precipitation with ammonium sulfate, PEG, antibodies and the like or by heat denaturation, followed by centrifugation; gel filtration, reverse phase, hydroxylapatite and affinity chromatography; and combinations of such and other techniques.
- polypeptide In purifying an antibody of the present invention, it may be desirable to express the polypeptide in a prokaryotic or eukaryotic expression system and extract the protein using denaturing conditions.
- the polypeptide may be purified from other cellular components using an affinity column, which binds to a tagged portion of the polypeptide.
- affinity column which binds to a tagged portion of the polypeptide.
- antibodies are fractionated utilizing agents (i.e., protein A) that bind the Fc portion of the antibody.
- agents i.e., protein A
- antigens my be used to simultaneously purify and select appropriate antibodies.
- Such methods often utilize the selection agent bound to a support, such as a column, filter or bead.
- the antibodies is bound to a support, contaminants removed (e.g., washed away), and the antibodies released by applying conditions (salt, heat, etc.).
- compositions comprising anti-AQP4 antibodies and antigens for generating the same.
- Such compositions comprise a prophylactically or therapeutically effective amount of an antibody or a fragment thereof, and a pharmaceutically acceptable carrier.
- pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
- carrier refers to a diluent, excipient, or vehicle with which the therapeutic is administered.
- Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a particular carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
- suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
- compositions can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
- These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like.
- Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical agents are described in "Remington's Pharmaceutical Sciences.”
- Such compositions will contain a prophylactically or therapeutically effective amount of the antibody or fragment thereof, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient.
- the formulation should suit the mode of administration, which can be oral, intravenous, intraarterial, intrabuccal, intranasal, nebulized, bronchial inhalation, or delivered by mechanical ventilation.
- Passive transfer of antibodies generally will involve the use of intravenous or intramuscular injections.
- the forms of antibody can be human or animal blood plasma or serum, as pooled human immunoglobulin for intravenous (IVIG) or intramuscular (IG) use, as high-titer human IVIG or IG from immunized or from donors recovering from disease, and as monoclonal antibodies (MAb).
- IVIG intravenous
- IG intramuscular
- MAb monoclonal antibodies
- Such immunity generally lasts for only a short period of time, and there is also a potential risk for hypersensitivity reactions, and serum sickness, especially from gamma globulin of non- human origin.
- passive immunity provides immediate protection.
- the antibodies will be formulated in a carrier suitable for injection, i.e., sterile and syringeable.
- compositions are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
- a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
- the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
- an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
- compositions can be formulated as neutral or salt forms.
- Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
- this treatment may involve administering to the patient the antibody of the present invention the other agent(s) at the same time.
- This may be achieved by use of a single pharmaceutical composition that includes both agents, or by administering two distinct compositions at the same time, wherein one composition includes the antibody of the present invention and the other includes the second agent(s).
- the two therapies may be given in either order and may precede or follow the other treatment by intervals ranging from minutes to weeks.
- the other agents are applied separately, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the agents would still be able to exert an advantageously combined effect on the patient.
- the antibody treatment of the present invention is "A” and the secondary treatment is "B":
- Corticosteroids high dose for acute attacks; low dose for chronic therapy
- prednisone are the main line therapy for NMO and thus are readily applied as a combined therapy with the anti-AQP4 antibodies of the present invention.
- Immunosuppressant treatments that can be used in combination with anti-AQP4 antibodies include azathioprine (Imuran) plus prednisone, mycophenolate mofetil plus prednisone, Rituximab, Mitoxantrone, intravenous immunoglobulin (IVIG), and cyclophosphamide.
- azathioprine Imuran
- prednisone mycophenolate mofetil plus prednisone
- Rituximab Mitoxantrone
- IVIG intravenous immunoglobulin
- cyclophosphamide ii. Plasmapheresis
- Plasmapheresis is the removal, treatment, and return of (components of) blood plasma from blood circulation. It is thus an extracorporeal therapy (a medical procedure which is performed outside the body). The method can also be used to collect plasma for further manufacturing into a variety of medications. The procedure is used to treat a variety of disorders, including those of the immune system, such as Myasthenia gravis, Guillain-Barre syndrome, lupus, thrombotic thrombocytopenic purpura and NMO.
- Plasmapheresis blood is initially taken out of the body through a needle or previously implanted catheter. Plasma is then removed from the blood by a cell separator. Three procedures are commonly used to separate the plasma from the blood cells:
- Discontinuous flow centrifugation One venous catheter line is required. Typically, a 300 ml batch of blood is removed at a time and centrifuged to separate plasma from blood cells. • Continuous flow centrifugation: Two venous lines are used. This method requires slightly less blood volume to be out of the body at any one time as it is able to continuously spin out plasma.
- Plasma filtration Two venous lines are used. The plasma is filtered using standard hemodialysis equipment. This continuous process requires less than 100 ml of blood to be outside the body at one time.
- each method has its advantages and disadvantages.
- the blood cells are returned to the person undergoing treatment, while the plasma, which contains the antibodies, is first treated and then returned to the patient in traditional plasmapheresis.
- the removed plasma is discarded and the patient receives replacement donor plasma, albumin, or a combination of albumin and saline (usually 70% albumin and 30% saline).
- replacement donor plasma albumin
- albumin or a combination of albumin and saline (usually 70% albumin and 30% saline).
- other replacement fluids such as hydroxyethyl starch, may be used in individuals who object to blood transfusion but these are rarely used due to severe side- effects.
- Medication to keep the blood from clotting an anticoagulant is given to the patient during the procedure.
- Plasma exchange therapy in and of itself is useful to temper the disease process, where simultaneous medical and immunosuppressive therapy is required for long-term management.
- Plasma exchange offers the quickest short-term answer to removing harmful autoantibodies; however, the production of autoantibodies by the immune system must also be suppressed, usually by the use of medications such as prednisone, cyclophosphamide, cyclosporine, mycophenolate mofetil, rituximab or a mixture of these.
- Antibodies may be linked to at least one agent to form an antibody conjugate.
- it is conventional to link or covalently bind or complex at least one desired molecule or moiety.
- a molecule or moiety may be, but is not limited to, at least one effector or reporter molecule.
- Effector molecules comprise molecules having a desired activity, e.g., immunosuppression/anti-inflammation. Non-limiting examples of such molecules are set out above.
- Such molecules are optionally attached via cleavable linkers designed to allow the molecules to be released at or near the target site.
- reporter molecule is defined as any moiety which may be detected using an assay.
- reporter molecules which have been conjugated to antibodies include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, photoaffinity molecules, colored particles or ligands, such as biotin.
- Antibody conjugates are generally preferred for use as diagnostic agents.
- Antibody diagnostics generally fall within two classes, those for use in in vitro diagnostics, such as in a variety of immunoassays, and those for use in vivo diagnostic protocols, generally known as "antibody-directed imaging.”
- Many appropriate imaging agents are known in the art, as are methods for their attachment to antibodies (see, for e.g., U.S. Patents 5,021,236, 4,938,948, and 4,472,509).
- the imaging moieties used can be paramagnetic ions, radioactive isotopes, fluorochromes, NMR-detectable substances, and X-ray imaging agents.
- paramagnetic ions such as chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) and/or erbium (III), with gadolinium being particularly preferred.
- Ions useful in other contexts, such as X-ray imaging include but are not limited to lanthanum (III), gold (III), lead (II), and especially bismuth (III).
- radioactive isotopes for therapeutic and/or diagnostic application, one might mention astatine 211 , 14 carbon, 51 chromium, 36 chlorine, 57 cobalt, 58 cobalt, copper 67 ,
- Radioactively labeled monoclonal antibodies may be produced according to well-known methods in the art.
- monoclonal antibodies can be iodinated by contact with sodium and/or potassium iodide and a chemical oxidizing agent such as sodium hypochlorite, or an enzymatic oxidizing agent, such as lactoperoxidase.
- Monoclonal antibodies may be labeled with technetium 991 " by ligand exchange process, for example, by reducing pertechnate with stannous solution, chelating the reduced technetium onto a Sephadex column and applying the antibody to this column.
- direct labeling techniques may be used, e.g., by incubating pertechnate, a reducing agent such as SNC1 2 , a buffer solution such as sodium- potassium phthalate solution, and the antibody.
- Intermediary functional groups which are often used to bind radioisotopes which exist as metallic ions to antibody are diethylenetriaminepentaacetic acid (DTP A) or ethylene diaminetetracetic acid (EDTA).
- fluorescent labels contemplated for use as conjugates include Alexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM, Fluorescein Isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red, Renographin, ROX, TAMRA, TET, Tetramethylrhodamine, and/or Texas Red.
- antibody conjugates contemplated are those intended primarily for use in vitro, where the antibody is linked to a secondary binding ligand and/or to an enzyme (an enzyme tag) that will generate a colored product upon contact with a chromogenic substrate.
- suitable enzymes include urease, alkaline phosphatase, (horseradish) hydrogen peroxidase or glucose oxidase.
- Preferred secondary binding ligands are biotin and avidin and streptavidin compounds. The use of such labels is well known to those of skill in the art and are described, for example, in U.S. Patents 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149 and 4,366,241.
- hapten-based affinity labels react with amino acids in the antigen binding site, thereby destroying this site and blocking specific antigen reaction.
- this may not be advantageous since it results in loss of antigen binding by the antibody conjugate.
- Molecules containing azido groups may also be used to form covalent bonds to proteins through reactive nitrene intermediates that are generated by low intensity ultraviolet light (Potter and Haley, 1983).
- 2- and 8-azido analogues of purine nucleotides have been used as site-directed photoprobes to identify nucleotide binding proteins in crude cell extracts (Owens & Haley, 1987; Atherton et ah, 1985).
- the 2- and 8-azido nucleotides have also been used to map nucleotide binding domains of purified proteins (Khatoon et ah, 1989; King et ah, 1989; Dholakia et ah, 1989) and may be used as antibody binding agents.
- Some attachment methods involve the use of a metal chelate complex employing, for example, an organic chelating agent such as diethylenetriaminepentaacetic acid anhydride (DTPA); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide; and/or tetrachloro-3a-6a-diphenylglycouril-3 attached to the antibody (U.S. Patents 4,472,509 and 4,938,948).
- DTPA diethylenetriaminepentaacetic acid anhydride
- ethylenetriaminetetraacetic acid N-chloro-p-toluenesulfonamide
- tetrachloro-3a-6a-diphenylglycouril-3 attached to the antibody
- Monoclonal antibodies may also be reacted with an enzyme in the presence of a coupling agent such as glutaraldehyde or periodate.
- Conjugates with fluorescein markers are prepared in the presence of these coupling agents or by reaction with an isothiocyanate.
- imaging of breast tumors is achieved using monoclonal antibodies and the detectable imaging moieties are bound to the antibody using linkers such as methyl-p- hydroxybenzimidate or N-succinimidyl-3 -(4-hydroxyphenyl)propionate.
- derivatization of immunoglobulins by selectively introducing sulfhydryl groups in the Fc region of an immunoglobulin, using reaction conditions that do not alter the antibody combining site are contemplated.
- Antibody conjugates produced according to this methodology are disclosed to exhibit improved longevity, specificity and sensitivity (U.S. Patent 5,196,066, incorporated herein by reference).
- Site-specific attachment of effector or reporter molecules, wherein the reporter or effector molecule is conjugated to a carbohydrate residue in the Fc region have also been disclosed in the literature (O'Shannessy et al., 1987). This approach has been reported to produce diagnostically and therapeutically promising antibodies which are currently in clinical evaluation.
- immunodetection methods for binding, purifying, removing, quantifying and otherwise generally detecting AQP4 and its associated antigens.
- Some immunodetection methods include enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradiometric assay, fluoroimmunoassay, chemiluminescent assay, bioluminescent assay, and Western blot to mention a few.
- ELISA enzyme linked immunosorbent assay
- RIA radioimmunoassay
- immunoradiometric assay fluoroimmunoassay
- fluoroimmunoassay chemiluminescent assay
- bioluminescent assay bioluminescent assay
- Western blot to mention a few.
- a competitive assay for the detection and quantitation of AQP4 antibodies also is provided.
- the immunobinding methods include obtaining a sample and contacting the sample with a first antibody in accordance with embodiments discussed herein, as the case may be, under conditions effective to allow the formation of immunocomplexes.
- the chosen biological sample with the antibody under effective conditions and for a period of time sufficient to allow the formation of immune complexes is generally a matter of simply adding the antibody composition to the sample and incubating the mixture for a period of time long enough for the antibodies to form immune complexes with, i.e., to bind to AQP4 present.
- the sample-antibody composition such as a tissue section, ELISA plate, dot blot or Western blot, will generally be washed to remove any non-specifically bound antibody species, allowing only those antibodies specifically bound within the primary immune complexes to be detected.
- the antibody employed in the detection may itself be linked to a detectable label, wherein one would then simply detect this label, thereby allowing the amount of the primary immune complexes in the composition to be determined.
- the first antibody that becomes bound within the primary immune complexes may be detected by means of a second binding ligand that has binding affinity for the antibody.
- the second binding ligand may be linked to a detectable label.
- the second binding ligand is itself often an antibody, which may thus be termed a "secondary" antibody.
- the primary immune complexes are contacted with the labeled, secondary binding ligand, or antibody, under effective conditions and for a period of time sufficient to allow the formation of secondary immune complexes.
- the secondary immune complexes are then generally washed to remove any non-specifically bound labeled secondary antibodies or ligands, and the remaining label in the secondary immune complexes is then detected.
- Further methods include the detection of primary immune complexes by a two step approach.
- a second binding ligand such as an antibody that has binding affinity for the antibody, is used to form secondary immune complexes, as described above.
- the secondary immune complexes are contacted with a third binding ligand or antibody that has binding affinity for the second antibody, again under effective conditions and for a period of time sufficient to allow the formation of immune complexes (tertiary immune complexes).
- the third ligand or antibody is linked to a detectable label, allowing detection of the tertiary immune complexes thus formed. This system may provide for signal amplification if this is desired.
- One method of immunodetection uses two different antibodies.
- a first biotinylated antibody is used to detect the target antigen, and a second antibody is then used to detect the biotin attached to the complexed biotin.
- the sample to be tested is first incubated in a solution containing the first step antibody. If the target antigen is present, some of the antibody binds to the antigen to form a biotinylated antibody/antigen complex.
- the antibody/antigen complex is then amplified by incubation in successive solutions of streptavidin (or avidin), biotinylated DNA, and/or complementary biotinylated DNA, with each step adding additional biotin sites to the antibody/antigen complex.
- the amplification steps are repeated until a suitable level of amplification is achieved, at which point the sample is incubated in a solution containing the second step antibody against biotin.
- This second step antibody is labeled, as for example with an enzyme that can be used to detect the presence of the antibody/antigen complex by histoenzymology using a chromogen substrate.
- a conjugate can be produced which is macroscopically visible.
- PCR Polymerase Chain Reaction
- the PCR method is similar to the Cantor method up to the incubation with biotinylated DNA, however, instead of using multiple rounds of streptavidin and biotinylated DNA incubation, the DNA/biotin/streptavidin/antibody complex is washed out with a low pH or high salt buffer that releases the antibody. The resulting wash solution is then used to carry out a PCR reaction with suitable primers with appropriate controls.
- the enormous amplification capability and specificity of PCR can be utilized to detect a single antigen molecule.
- Immunoassays in their most simple and direct sense, are binding assays. Certain preferred immunoassays are the various types of enzyme linked immunosorbent assays (ELISAs) and radioimmunoassays (RIA) known in the art. Immunohistochemical detection using tissue sections is also particularly useful. However, it will be readily appreciated that detection is not limited to such techniques, and western blotting, dot blotting, FACS analyses, and the like may also be used.
- the antibodies of the invention are immobilized onto a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. Then, a test composition suspected of containing the AQP4 is added to the wells. After binding and washing to remove non-specifically bound immune complexes, the bound antigen may be detected. Detection may be achieved by the addition of another anti-AQP4 antibody that is linked to a detectable label.
- ELISA is a simple "sandwich ELISA.” Detection may also be achieved by the addition of a second anti-AQP4 antibody, followed by the addition of a third antibody that has binding affinity for the second antibody, with the third antibody being linked to a detectable label.
- the samples suspected of containing the AQP4 antigen are immobilized onto the well surface and then contacted with anti-AQP4 antibody. After binding and washing to remove non-specifically bound immune complexes, the bound anti- AQP4 antibodies are detected. Where the initial anti-AQP4 antibodies are linked to a detectable label, the immune complexes may be detected directly. Again, the immune complexes may be detected using a second antibody that has binding affinity for the first anti-AQP4 antibody, with the second antibody being linked to a detectable label.
- ELISAs have certain features in common, such as coating, incubating and binding, washing to remove non-specifically bound species, and detecting the bound immune complexes. These are described below.
- a plate with either antigen or antibody In coating a plate with either antigen or antibody, one will generally incubate the wells of the plate with a solution of the antigen or antibody, either overnight or for a specified period of hours. The wells of the plate will then be washed to remove incompletely adsorbed material. Any remaining available surfaces of the wells are then "coated" with a nonspecific protein that is antigenically neutral with regard to the test antisera. These include bovine serum albumin (BSA), casein or solutions of milk powder.
- BSA bovine serum albumin
- the coating allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface.
- a secondary or tertiary detection means rather than a direct procedure.
- the immobilizing surface is contacted with the biological sample to be tested under conditions effective to allow immune complex (antigen/antibody) formation. Detection of the immune complex then requires a labeled secondary binding ligand or antibody, and a secondary binding ligand or antibody in conjunction with a labeled tertiary antibody or a third binding ligand.
- Under conditions effective to allow immune complex (antigen/antibody) formation means that the conditions preferably include diluting the antigens and/or antibodies with solutions such as BSA, bovine gamma globulin (BGG) or phosphate buffered saline (PBS)/Tween. These added agents also tend to assist in the reduction of nonspecific background.
- the "suitable” conditions also mean that the incubation is at a temperature or for a period of time sufficient to allow effective binding. Incubation steps are typically from about 1 to 2 to 4 hours or so, at temperatures preferably on the order of 25°C to 27°C, or may be overnight at about 4°C or so.
- the contacted surface is washed so as to remove non-complexed material.
- a preferred washing procedure includes washing with a solution such as PBS/Tween, or borate buffer. Following the formation of specific immune complexes between the test sample and the originally bound material, and subsequent washing, the occurrence of even minute amounts of immune complexes may be determined.
- the second or third antibody will have an associated label to allow detection.
- this will be an enzyme that will generate color development upon incubating with an appropriate chromogenic substrate.
- a urease, glucose oxidase, alkaline phosphatase or hydrogen peroxidase-conjugated antibody for a period of time and under conditions that favor the development of further immune complex formation (e.g., incubation for 2 hours at room temperature in a PBS-containing solution such as PBS-Tween).
- the amount of label is quantified, e.g., by incubation with a chromogenic substrate such as urea, or bromocresol purple, or 2,2'-azino-di-(3-ethyl-benzthiazoline-6- sulfonic acid (ABTS), or ⁇ 2 0 2 , in the case of peroxidase as the enzyme label. Quantification is then achieved by measuring the degree of color generated, e.g., using a visible spectra spectrophotometer. 2.
- the Western blot is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein.
- a membrane typically nitrocellulose or PVDF
- Samples may be taken from whole tissue or from cell culture. In most cases, solid tissues are first broken down mechanically using a blender (for larger sample volumes), using a homogenizer (smaller volumes), or by sonication. Cells may also be broken open by one of the above mechanical methods. However, it should be noted that bacteria, virus or environmental samples can be the source of protein and thus Western blotting is not restricted to cellular studies only. Assorted detergents, salts, and buffers may be employed to encourage lysis of cells and to solubilize proteins. Protease and phosphatase inhibitors are often added to prevent the digestion of the sample by its own enzymes. Tissue preparation is often done at cold temperatures to avoid protein denaturing.
- the proteins of the sample are separated using gel electrophoresis. Separation of proteins may be by isoelectric point (pi), molecular weight, electric charge, or a combination of these factors. The nature of the separation depends on the treatment of the sample and the nature of the gel. This is a very useful way to determine a protein. It is also possible to use a two-dimensional (2-D) gel which spreads the proteins from a single sample out in two dimensions. Proteins are separated according to isoelectric point (pH at which they have neutral net charge) in the first dimension, and according to their molecular weight in the second dimension.
- isoelectric point pH at which they have neutral net charge
- the proteins In order to make the proteins accessible to antibody detection, they are moved from within the gel onto a membrane made of nitrocellulose or polyvinylidene difluoride (PVDF).
- PVDF polyvinylidene difluoride
- the membrane is placed on top of the gel, and a stack of filter papers placed on top of that. The entire stack is placed in a buffer solution which moves up the paper by capillary action, bringing the proteins with it.
- Another method for transferring the proteins is called electroblotting and uses an electric current to pull proteins from the gel into the PVDF or nitrocellulose membrane.
- the proteins move from within the gel onto the membrane while maintaining the organization they had within the gel. As a result of this blotting process, the proteins are exposed on a thin surface layer for detection (see below).
- the antibodies may also be used in conjunction with both fresh-frozen and/or formalin-fixed, paraffin-embedded tissue blocks prepared for study by immunohistochemistry (IHC).
- IHC immunohistochemistry
- frozen-sections may be prepared by rehydrating 50 ng of frozen "pulverized” tissue at room temperature in phosphate buffered saline (PBS) in small plastic capsules; pelleting the particles by centrifugation; resuspending them in a viscous embedding medium (OCT); inverting the capsule and/or pelleting again by centrifugation; snap-freezing in -70°C isopentane; cutting the plastic capsule and/or removing the frozen cylinder of tissue; securing the tissue cylinder on a cryostat microtome chuck; and/or cutting 25-50 serial sections from the capsule.
- whole frozen tissue samples may be used for serial section cuttings.
- Permanent-sections may be prepared by a similar method involving rehydration of the 50 mg sample in a plastic micro fug e tube; pelleting; resuspending in 10% formalin for 4 hours fixation; washing/pelleting; resuspending in warm 2.5% agar; pelleting; cooling in ice water to harden the agar; removing the tissue/agar block from the tube; infiltrating and/or embedding the block in paraffin; and/or cutting up to 50 serial permanent sections. Again, whole tissue samples may be substituted.
- immunodetection kits for use with the immunodetection methods described above.
- the immunodetection kits will thus comprise, in suitable container means, a first antibody that binds to AQP4 antigen, and optionally an immunodetection reagent.
- the AQP4 antibody may be pre-bound to a solid support, such as a column matrix and/or well of a microtitre plate.
- the immunodetection reagents of the kit may take any one of a variety of forms, including those detectable labels that are associated with or linked to the given antibody. Detectable labels that are associated with or attached to a secondary binding ligand are also contemplated. Exemplary secondary ligands are those secondary antibodies that have binding affinity for the first antibody.
- Further suitable immunodetection reagents for use in the present kits include the two- component reagent that comprises a secondary antibody that has binding affinity for the first antibody, along with a third antibody that has binding affinity for the second antibody, the third antibody being linked to a detectable label.
- a number of exemplary labels are known in the art and all such labels may be employed in connection with embodiments discussed herein.
- kits may further comprise a suitably aliquoted composition of the AQP4 antigen, whether labeled or unlabeled, as may be used to prepare a standard curve for a detection assay.
- the kits may contain antibody-label conjugates either in fully conjugated form, in the form of intermediates, or as separate moieties to be conjugated by the user of the kit.
- the components of the kits may be packaged either in aqueous media or in lyophilized form.
- the container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which the antibody may be placed, or preferably, suitably aliquoted.
- the kits will also include a means for containing the antibody, antigen, and any other reagent containers in close confinement for commercial sale.
- Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.
- EXAMPLE 1 MATERIALS AND METHODS DNA constructs, cell culture and transfections.
- DNA constructs encoding full-length human AQP4 (Ml and M23 iso forms) were generated by PCR-amplification using whole brain cDNA as template.
- a Myc epitope (NH 2 -EQKLISEEDL-COOH; SEQ ID NO: 13) was inserted in the second extracellular loop by PCR-amplification using the non-tagged constructs as template.
- Mutants of Ml and M23 were generated by PCR-amplification using either tagged or non-tagged templates. All PCR fragments were ligated into mammalian expression vector pcDNA3.1 and fully sequenced.
- U87MG cell cultures (ATCC HTB-14) were maintained at 37 °C in 5% C0 2 /95% air in EMEM medium containing 10% fetal bovine serum, 100 U/mL penicillin and 100 ⁇ g/mL streptomycin. Cells were grown on glass coverslips and transfected with DNA in antibiotic-free medium using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. Stable AQP4-expressing clones were selected following enrichment in Geneticin (Invitrogen) and plating in 96-well plates at very low density.
- NMO patient sera and recombinant AQP4 autoantibodies were obtained from four NMO-IgG seropositive individuals who met the revised diagnostic criteria for clinical disease (Wingerchuk et ah, 2006).
- Control (non-NMO) human serum was purchased from the UCSF cell culture facility.
- Recombinant monoclonal NMO antibodies were generated from clonally-expanded cerebrospinal fluid plasma blasts as described previously (Bennett et ah, 2009). Heavy- and light-chain constructs were co-transfected into HEK293 cells, the supernatant harvested, centrifuged to remove any cells and debris, and incubated overnight with protein A- Sepharose (Sigma- Aldrich, St.
- the rAb was eluted in 0.1 M glycine / 1 M NaCl (pH 3.0) and adjusted to pH 7.5 with 0.1 M Tris-HCl, pH 8.0.
- Recombinant IgG was subsequently exchanged and concentrated in PBS containing 0.1% protease-free bovine serum albumin using Ultracel YM-30 microconcentrators (Millipore, Billerica, MA).
- Fab fragments were generated by digestion of whole IgG with immobilized papain, and purified by removal of undigested IgG and Fc fragments by protein-A (Thermo Fisher Scientific, Rockford, IL). Antibody integrity was confirmed by denaturing and native gel electrophoresis, and IgG concentration was assayed using a human IgG-capture ELISA.
- AQP4-expressing U87MG cells were incubated for 20 min in live-cell blocking buffer (PBS containing 6 mM glucose, 1 mM pyruvate, 1 % bovine serum albumin, 2% goat serum), and then for 30 min with NMO patient serum or recombinant NMO-IgG in blocking buffer. Cells were then rinsed extensively with PBS, fixed in 4% paraformaldehyde for 15 min, and permeabilized with 0.1% Triton X-100.
- live-cell blocking buffer PBS containing 6 mM glucose, 1 mM pyruvate, 1 % bovine serum albumin, 2% goat serum
- U87MG cells were labeled as described above, but with a monoclonal mouse anti-Myc IgG (Covance, Emeryville, CA) or purified Fab fragments instead of whole NMO-IgG.
- Anti-Myc was stained with goat anti-mouse IgG-conjugated Alexa Fluor 488 (Invitrogen), while Fab fragments were stained with Dylight 488-linked F(ab') 2 -specific secondary antibodies (Jackson Immunoresearch, West Grove, PA).
- Quantitative analysis of AQP4-antibody binding was done on a Nikon Eclipse TE2000S inverted epifluorescence microscope (Nikon, Melville, NY) equipped with a Nikon lOx air objective (numerical aperture 0.3). Green and red dyes were excited and observed through Chroma filter sets #41001 and #42001 (Chroma, Rockingham, VT), respectively. Images were recorded by a CCD camera (Hamamatsu Orca, Bridgewater, NJ), and intensities determined using custom software.
- TIRFM Total internal reflection fluorescence microscopy.
- TIRFM was done using a Nikon Eclipse TE2000E microscope with a through-objective TIRF attachment and a lOOx TIRF oil immersion objective (numerical aperture 1.49) mounted on a perfect focus module (Nikon).
- Alexa Fluor 555-labeled AQP4 was excited using an argon-ion laser through a Z514/1 Ox excitation filter and Z514RDC dichroic mirror, and detected through an ET605/40m emission filter (Chroma). Images were acquired using a QuantEM 512SC deep-cooled CCD camera (Photometries, Arlington, AZ).
- SPT was performed on a Nikon Eclipse TE2000S inverted epifluorescence microscope equipped with a Nikon lOOx TIRF oil immersion objective (numerical aperture 1.45) and a deep-cooled CCD camera (Hamamatsu EM-CCD, Bridgewater, NJ).
- Qdot fluorescence was excited using an E460SPUV excitation filter and 475DCXRU dichroic mirror, and detected through a D655/40m emission filter (Chroma). Data were acquired continuously at 11 ms per frame (91 Hz) for 6 s. Image sequences were analyzed and trajectories constructed as described in detail previously (Crane and Verkman, 2008).
- Electrophoresis and immunoblotting Cell cultures were lysed with NativePAGE sample buffer (Invitrogen) containing 0.5 % dodecyl- -D-maltoside (EMD chemicals, Gibbstown, NJ) for 10 min on ice. Lysates were centrifuged at 20,000 g for 30 min at 4 °C and the pellet discarded.
- NativePAGE sample buffer Invitrogen
- EMD chemicals Gibbstown, NJ
- polyacrylamide native gradient gels (3-9 %) were prepared as previously described (Wittig et ah, 2006). 10 ⁇ g of protein was mixed with 5% Coomassie Blue G-250 (Invitrogen) and loaded in each lane. Ferritin was used as the molecular mass standard (440 and 880 kDa). Running buffers were: 25 mM imidazole, pH 7 (anode buffer) and 50 mM Tricine, 7.5 mM imidazole, 0.02% Coomassie Blue G-250, pH 7 (cathode buffer).
- Tricine SDS-PAGE was performed as previously described (Schagger and Tricine, 2006) with a 12% running gel and 3% stacking gel. Samples were not heated prior to loading. SeeBlue Plus2 Pre-Stained Standard (Invitrogen) was used as a molecular weight marker. Proteins were blotted onto polyvinyl difluoride membranes (Bio-Rad, Hercules, CA). For immunoblot analysis, membranes were blocked with 3% BSA and incubated with rabbit anti-AQP4 primary antibodies (Santa Cruz) for 2 h.
- Membranes were then rinsed and incubated for 1 h with horseradish peroxidase-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch), rinsed extensively, and labeled proteins were detected using the ECL Plus enzymatic chemiluminescence kit (Amersham Biosciences, Pittsburgh, PA).
- FIG. 1 diagrams the approach used for quantitative analysis of NMO-IgG binding to AQP4.
- Cells expressing specified AQP4 isoform(s) at their plasma membrane were incubated with NMO- IgG (NMO patient serum or recombinant monoclonal antibody), fixed, permeabilized, and then incubated with anti-AQP4 antibody (FIG. 1 A).
- the anti-AQP4 antibody recognizes the AQP4 C-terminus, which is common to all AQP4 isoforms (FIG. IB).
- Fluorescent secondary antibodies were used to detect NMO-IgG by green fluorescence and AQP4 by red fluorescence.
- NMO-IgG binding to AQP4 was quantified by green-to-red (G/R) fluorescence ratio. Binding affinity and stoichometry was determined by titration with increasing NMO- IgG concentration. In contrast to measurements done at a single NMO-IgG concentration, measurement of full, concentration-dependent binding provides a quantitative, unbiased description of NMO-IgG binding to AQP4.
- G/R green-to-red
- FIG. 2A shows high-magnification confocal microscopy of U87MG cells stably expressing Ml or M23 AQP4, and stained with a recombinant monoclonal NMO-IgG (green) and an anti-AQP4 antibody (red).
- FIG. 2B shows TIRFM of the anti-AQP4 antibody-stained AQP4-expressing cells.
- a smooth pattern of fluorescence was seen for M1-AQP4 and a punctate pattern for M23-AQP4, as found in other cell types (Crane et al., 2008), confirming that M23-AQP4 forms OAPs in transfected U87MG cells whereas M1-AQP4 does not.
- Immunoblot analysis of cell homogenates showed the expected molecular sizes of the Ml and M23 iso forms of AQP4 (FIG. 2C, bottom).
- FIG. 2D shows examples of measured green-to- red ratios (G/R) after binding recombinant monoclonal NMO-IgGs (rAb-53, rAb-58, and rAb-186) or control antibody (rAb-2B4) (each at 20 ⁇ g/mL) to Ml or M23 AQP4-expressing cells.
- FIG. 3A shows representative fluorescence micrographs for serum specimens from four NMO patients. Each serum specimen showed strong NMO-IgG binding to M23 AQP4, but variable binding to Ml AQP4.
- FIG. 3B shows concentration- dependent NMO-IgG binding in which background-corrected R/G ratios were determined as a function of serum concentration.
- NMO-IgG binding to AQP4 was saturable, with relative M23:M1 binding affinities of 0.11 (serum 1), 0.26 (serum 2), 0.03 (serum 3) and 0.21 (serum 4).
- the binding data fitted well to a single-site, saturable binding model with near unity Hill coefficient, despite the presumed polyclonal composition of NMO serum. It is not possible to determine absolute binding affinity of NMO-IgG to AQP4 using serum because the fraction of NMO-lgG in total serum IgG is not known, nor is the polyclonal NMO-lgG composition.
- NMO-lgG in serum from a single NMO patient is polyclonal, consisting of a pool of monoclonal NMO-IgGs.
- a similar binding analysis was done using monoclonal recombinant NMO-IgGs derived from a single NMO patient in order to determine: (i) absolute binding affinities; (ii) whether differential M23:M1 is heterogeneous in a single NMO patient; and (iii) whether differential M23:M1 binding is due to differences in NMO-lgG binding affinity and/or stoichiometry.
- FIG. 4A shows fluorescence micrographs of two recombinant monoclonal NMO-IgGs (rAb-53 and rAb-58) from a single NMO patient.
- the monoclonal antibodies used in this study were derived from the CSF of a single NMO patient (corresponding to 'serum in FIG. 3). Though strong monoclonal antibody binding to M23 AQP4 was seen in each case, binding to Ml AQP4 was variable.
- FIG. 4B summarizes concentration-dependence binding curves for three recombinant NMO-IgGs, together with curve fits for a single-site binding model. In each case the data fitted well to a single-site model with near unity Hill coefficient.
- the lowest dissociation constant was 44 nM, which was found for binding of rAb-53 to M23 AQP4. Marked heterogeneity was found for monoclonal NMO-IgGs from a single NMO patient, with relative M23:M1 binding affinities of 0.02 (rAb-53), 0.46 (rAb-58) and 0.02 (rAb-186).
- the binding curves in FIG. 4B also support the conclusion that differences in M23:M1 binding are due to differences in binding affinity rather than to binding capacity.
- Mechanism of AQP4 isoform-specific binding of NMO-lgG The inventors investigated whether differences in NMO-lgG binding affinity to M23 vs. Ml AQP4 are due to formation of OAPs by M23 AQP4 and/or to the different N-termini of M23 vs. Ml . Measurements were made of NMO-lgG binding to cells expressing: (i) different ratios of M23:M1 AQP4; (ii) an Ml mutant that has a diminished ability to disrupt OAPs when coexpressed with M23; and (iii) M23 AQP4 mutants that have diminished ability to form OAPs. For these studies, the inventors used transiently transfected U87MG cells. The suitability of transiently transfected cells was validated above by showing comparable binding in stably vs. transiently transfected cells by NMO-lgG at a fixed concentration (FIG. 2D).
- FIG. 5A shows concentration-dependent binding of NMO-lgG (rAb-53) to U87MG cells co-expressing different ratios of M23:M1 AQP4.
- the fraction of AQP4 in OAPs and average OAP size are directly related to the M23:M1 AQP4 ratio.
- Quantum dot single- particle tracking measurements were done to determine the characteristics of OAPs formed at different M23:M1 ratios.
- FIG. 5A (right) shows increased AQP4 diffusion with higher Ml content, as previously shown (Crane et al., 2009). This increase in AQP4 diffusion is due to active disruption of OAP growth by Ml AQP4.
- rAb-53 showed incrementally reduced AQP4 binding in 3: 1 and 1 : 1 mixtures of M23:M1 (FIG. 5 A, left).
- FIG. 5B shows data from similar experiments as in FIG. 5 A, except that native Ml was substituted by the double-cysteine mutant M1-C13A/C17A (CCA).
- CCA AQP4 does not form OAPs on its own, but when co-expressed with M23 has greatly reduced ability to disrupt OAPs (Crane et al, 2009). Therefore, at the same M23:M1 ratio, cells expressing the CCA mutant in place of native Ml have greater OAP content, as confirmed by single particle tracking (FIG. 5B, right). Concentration-dependent binding of rAb-53 showed greater binding to M23:CCA mixtures than to M23:M1 mixtures (FIG. 5B, left). Binding of rAb-53 to cells expressing a 3: 1 ratio of M23:CCA was identical to cells expressing M23 alone.
- FIG. 5C shows that mutations F26Q and G28P in the M23 AQP4 N-terminus greatly reduce OAP content, similar to the inventors' previous findings with corresponding rat isoforms of AQP4 (Crane and Verkman, 2009).
- FIG. 5C shows greatly reduced concentration-dependent binding of rAb-53 to cells expressing these M23 mutants when compared to native M23.
- FIG. 6 A shows a comparison of the distance between adjacent Fab binding sites in IgGi (Sosnick et al, 1992; Harris et al, 1998), and the size of the AQP4 tetramer (Ho et al, 2009).
- FIG. 6B diagrams possible opposing binding mechanisms.
- First is bivalent binding. Strong NMO- IgG binding requires a bivalent interaction in which both Fab sites must bind to AQP4 monomers or tetramers.
- the positions of the binding epitopes in AQP4 monomers are spaced such that a bivalent interaction between the Fab sites is not possible within a single tetramer, but is optimal for crosslinking of adjacent tetramers in OAPs.
- the epitopes may be located at positions in which bivalent binding in a single tetramer can occur, resulting in similar binding to Ml and M23 AQP4.
- Second is monovalent binding. NMO-IgG binding involves classical monovalent interactions, and is controlled primarily by affinities of individual Fab's to their respective epitopes.
- rAb-53 a structural change in the epitope site upon OAP formation results in higher affinity.
- rAb-58 the epitope site is not altered by OAP formation, resulting in similar affinity for Ml vs. M23 AQP4.
- Fab binding to Ml and M23 AQP4 was measured to test these competing mechanisms.
- NMO-IgGs were digested with papain and purified to yield Fab's.
- the bivalent binding mechanism predicts little binding by Fab's, and no difference between Fab binding to Ml vs. M23 AQP4.
- the monovalent binding mechanism predicts that the increased binding of rAb-53 to M23 AQP4 would also be observed for its individual Fab's.
- the inventors measured binding of Fab's generated from a mouse monoclonal anti-Myc antibody to external Myc-tagged AQP4 isoforms.
- FIG. 6C shows the binding of Fab fragments to Ml vs. M23 AQP4.
- Whole IgG and Fab's from rAb- 53 showed significantly greater binding to M23 AQP4, while whole IgG and Fab's from rAb- 58 and anti-Myc showed similar Ml vs. M23 binding.
- the inventors used fluorescence ratio imaging to quantify the binding of NMO- IgG to AQP4, and to determine the role of AQP4 isoforms and OAPs in NMO-IgG binding.
- This live-cell system was developed out of a need for a robust method to characterize monoclonal NMO-IgGs and polyclonal NMO patient sera.
- the inventors found that U87MG cells were suitable for quantitative binding measurements because they efficiently expressed AQP4 at the plasma membrane after stable or transient transfection, with little or no intracellular AQP4 expression.
- the excellent membrane expression of AQP4 in U87MG cells is likely due to their glial origin, and hence their expression of the same trafficking machinery as that in native human glial cells.
- NMO-IgG was found to bind to each AQP4 isoform in a concentration-dependent manner that fitted well to a single-site, saturable binding model with near unity Hill coefficient, consistent with apparent single-site, non- cooperative binding.
- Preferential binding of NMO-IgG to M23 AQP4 was found to be a consequence of greater binding affinity rather than to greater binding capacity.
- Differences in the binding affinity of monoclonal NMO-IgG rAb-53 to the M23 and Ml isoforms of AQP4 was found to be a consequence of OAP formation by M23 AQP4 rather than to differences in the N-terminal sequences.
- binding to AQP4 mutants M23-F26Q and M23-G28P produced isotherms that did that not fit to a single-site binding model, probably because expression of these mutants produces heterogeneous populations of OAP-assembled AQP4 and non-assembled AQP4 tetramers.
- NMO-IgG binding was examined by comparing whole (bivalent) NMO-IgG to its monovalent Fab's. OAP assembly resulted in a greater affinity for individual Fab binding sites, rather than enhancement by bivalent IgG binding.
- the widely used assay for serum NMO-lgG immunofluorescence is performed in Ml AQP4-expressing HEK-293 cells (Lennon et al., 2005).
- the data here indicate that M23 AQP4-expressing cells are superior because NMO-lgG binding to M23 AQP4 is as good as and generally much better than binding to Ml AQP4.
- the difference in binding is seen at all NMO-lgG concentrations and is often quite marked at low concentrations, as often found in human serum specimens.
- Up to 30% of serum from patients with NMO, as defined by established clinical criteria, are found to be seronegative as assayed using the conventional method (Wingerchuk et al., 2006).
- This value is likely substantially lower utilizing more sensitive assays such as the imaging assay established here utilizing human glial cell line strongly expressing the OAP-forming M23 isoform of AQP4. Indeed, a recent study of multiple human serum samples indicated an improvement from 70% to 97% sensitivity for NMO-lgG when using M23 -expressing cells instead of Ml -expressing cells (Mader et al., 2010).
- This quantitative binding assay established here should also be useful in correlating serum NMO-lgG binding affinity and specificity with NMO clinical parameters, such as disease activity, treatment status and patient characteristics. Baseline differences in NMO- lgG binding to OAP vs. non-OAP associated AQP4 may have diagnostic significance, and spontaneous and treatment-associated changes in binding may have prognostic significance.
- Recombinant NMO-IgGs and NMO patient sera Recombinant monoclonal NMO antibodies (rAbs) were generated from clonally-expanded plasma blasts in cerebrospinal fluid (CSF) as described (Bennett et al., 2009).
- CSF cerebrospinal fluid
- Kpnl-Xhol fragment of the IgGl heavy chain constant region (IgGlFc) was cloned into pSp73X.
- Point mutations were introduced into the IgGlFc sequence using the GeneTailor Site-Directed Mutagenesis System (Invitrogen) to generate constructs deficient in CDC (mutation K322A), ADCC (mutations K326W/E333S) or both (mutations L234A/L235A) (Baudino et al, 2008; Duncan and Winter, 1988; Hezareh et al., 2001; Idusogie et al., 2001).
- the Agel-Xhol fragment of the mutated IgGlFc sequence was cloned into plgGlFlag containing the heavy- chain variable region sequence of rAb-53 to generate aquaporumab constructs containing a mutant IgGl Fc sequence with a C-terminal Flag epitope.
- paired heavy and light chain constructs were co-transfected into HEK293 cells using lipofectamine. After transfection, cells were grown for 7 days in DMEM medium + 10% FBS, the supernatant harvested, fresh medium added, and the cells were grown for another 7 days. The cell culture supernatants were combined, centrifuged at 2000 rpm for 10 min to pellet cells and debris, and the cell- free supernatant was adjusted to pH 8.0 with 1M Tris pH 8.0 and incubated overnight with protein A-Sepharose (Sigma- Aldrich) at 4 °C.
- the rAb was eluted from the protein A- Sepharose in 0.1 M glycine/lM NaCl (pH 3.0) and adjusted to pH 7.5 with 0.1M Tris-HCl, pH 8.0. Recombinant IgGs were exchanged and concentrated in PBS containing 0.1% protease-free bovine serum albumin (BSA) using Ultracel YM-30 microconcentrators (Millipore, Billerica, MA). BSA was excluded from the storage solution for surface plasmon resonance measurements. Antibody integrity was confirmed by denaturing and native gel electrophoresis, and IgG concentration was assayed using a human IgG-capture ELISA.
- BSA protease-free bovine serum albumin
- NMO serum was obtained from a total of ten NMO-IgG seropositive individuals who met the revised diagnostic criteria for clinical disease (Wingerchuk et al., 2006).
- Control (non-NMO) human serum was obtained from a total of three non-NMO individuals, or purchased from the UCSF cell culture facility.
- total IgG was purified and concentrated from serum using a Melon Gel IgG Purification Kit (Thermo Fisher Scientific, Rockford, IL) and Amicon Ultra Centrifugal Filter Units (Millipore, Billerica, MA).
- NK-92 cells expressing CD16 were cultured at 37 °C in 5% C0 2 /95%> air in a-MEM with deoxyribonucleosides and ribonucleosides containing 10% fetal bovine serum, 10% horse serum, 0.1 mM 2-mercaptoethanol, 2 mM glutamine, 0.2 mM myo-inositol, 2.5 ⁇ folic acid, non-essential amino acid, 110 ⁇ g/mL sodium pyruvate, 100 U/mL penicillin and 100 ⁇ g/mL streptomycin.
- lipids total 37.5 mg, 500 nmol
- lipids total 37.5 mg, 500 nmol
- lipids dissolved in 375 ⁇ , of 40 mM b-octyl glucoside (in PBS)
- AQP4 300 ml, 450 mg, 15 nmol
- the mixture was dialyzed (10,000 dalton cut-off) against PBS at 4 °C for 48 h.
- an equal volume of 40 mM b-octyl glucoside (instead of AQP4) was added to the lipid solution.
- Proteoliposomes were immobilized on a LI sensor chip (Biacore) with four injections at 10 min/injection at 2 ⁇ /min to achieve 6000 response units of proteoliposomes immobilization. The surface was then washed with two 20 s injections of 50 mM NaOH at 100 ⁇ /min, and checked for surface quality with a 300 s injection of 0.01 mg/ml BSA at 20 ⁇ /min.
- Flow channel 1 (Fc 1) was immobilized with 0% AQP4 as reference, and Fc 2 contained the AQP4 proteoliposomes. Binding studies were conducted with PBS at a flow rate of 15 ⁇ /min.
- rAbs in PBS were injected for 80 s followed by a post-injection period of 240 s. Regeneration was performed by injection of 50 mM NaOH at 100 ⁇ /min for 20 s. Binding studies were done in duplicate. Data were analyzed using Biacore T100 Evaluation software.
- NMO-IgG binding to AQP4 in cells The kinetics of rAb-53 binding to AQP4 was measured in U87MG cells stably expressing human AQP4-M23 by quantitative imaging as described (Crane et ah, 2011). Cells were incubated for 20 min in live-cell blocking buffer (PBS containing 6 mM glucose, 1 mM pyruvate, 1% bovine serum albumin, 2% goat serum), and then for specified times with NMO-IgG. Cells were then rinsed, fixed in 4 % paraformaldehyde for 15 min, and permeabilized with 0.1% Triton X-100.
- live-cell blocking buffer PBS containing 6 mM glucose, 1 mM pyruvate, 1% bovine serum albumin, 2% goat serum
- CDC complement-dependent cytotoxicity
- ADCC antibody-dependent cell-mediated cytotoxicity
- CHO cells expressing human AQP4 were pre-incubated for 30 min with 12.5 ⁇ g/ml aquaporumab (or control IgG), then for 90 min at 37 °C with NMO-IgG (2.5 ⁇ g/ml) or control-IgG and 5% human complement.
- Calcein-AM and ethidium-homodimer Invitrogen
- Complement-mediated cytotoxicity by 1-2% NMO patient sera (and control non-NMO sera) and 5% human complement were was measured similarly without vs.
- NK-92 cells expressing CD 16 were used as the effector cells.
- the AQP4-expressing CHO cells were pre -incubated for 30 min with 15 ⁇ / ⁇ 1 aquaporumab (or control-IgG), then for 3 h at 37 °C with NMO-IgG (5 ⁇ g/ml) or control-IgG and effector cells (effector : target ratio 30: 1). Cells were rinsed with PBS before adding calcein-AM and ethidium-homodimer.
- mice In vivo NMO model.
- Adult mice (30-35g) were anaesthetized with 2,2,2- tribromoethanol (125 mg/kg i.p.) and mounted in a stereotactic frame. Mice were injected intracerebrally, as described (Saadoun et ah , 2010), with purified total IgG (14 ⁇ ,, 6-38 mg/mL) isolated NMO patient serum (5 different NMO seropositive patients studied) plus human complement (10 ⁇ ), without or with aquaporumab (10 ⁇ g). Controls included non- NMO human IgG (3 different control sera studied), use of AQP4 null mice, and injection of aquaporumab alone.
- mice were killed at 24 h after injection and brains were fixed in formalin, processed into paraffin, and sectioned coronally at 1.6 mm from the frontal poles. Sections were stained with hematoxylin/eosin, Luxol Fast Blue (for myelin) or immunostained with antibodies against AQP4 (Millipore, Watford, UK) and C5b-9 (Abeam, Cambridge, UK). Micrographs were quantified for loss of AQP4 immunoreactivity and myelin as described (Saadoun et ah, 2010; Crane et ah , 201 1).
- Slices were cultured in 5% C0 2 at 37 °C for 10 days in 50% minimum essential medium (MEM), 25% HBSS, 25% horse serum, 1% penicillin-streptomycin, glucose (0.65%) and HEPES (25 mM) (changed every 3 days).
- MEM minimum essential medium
- HBSS 25% HBSS
- horse serum 1% penicillin-streptomycin
- glucose 0.65%
- HEPES 25 mM
- Sections were scores as follows: 0, intact slice with uniform and intact GFAP and AQP4 staining; 1 , intact slice with some astrocyte swelling seen by GFAP staining, with reduced AQP4 staining; 2, at least one lesion in the slice with loss of GFAP and AQP4 staining; 3, multiple lesions affecting > 30% of slice area with loss of GFAP and AQP4 staining; 4, extensive loss of GFAP and AQP4 staining affecting > 80% of slice area.
- Slices from AQP4 null mice scored from GFAP immunofluorescence only.
- FIG. 7A The rationale for aquaporumab therapy of NMO is depicted in FIG. 7A.
- Pathogenic autoantibodies that bind to extracellular epitopes on AQP4 are substantially larger than AQP4 tetramers, preventing the simultaneous binding of more than one antibody.
- the inventors reasoned, therefore, that a non-pathogenic antibody with high binding affinity and slow washout would compete with the binding of pathogenic antibodies and thus block downstream astrocyte damage and neuroinflammation.
- the inventors generated and screened ten recombinant monoclonal NMO-IgGs that were derived from clonally expanded plasma blast populations in the CSF of three NMO patients. Paired heavy and light chain variable region sequences from single cells were PCR-amp lifted, cloned into expression vectors containing heavy and light chain constant region sequences, coexpressed in HEK293 cells, and the recombinant IgG purified from supernatants. Binding of each monoclonal recombinant antibody to AQP4 in reconstituted proteoliposomes was measured by surface plasmon resonance.
- FIG. 7B shows highest affinity and slowest washout for antibody rAb-53 (FIG. 7B, left). Binding of rAb-53 to AQP4-proteoliposomes occurred within a few minutes (binding rate constant 1.4 x 10 4 M " V 1 ) and washout over many hours (off rate constant 3.8 x 10 "4 s "1 ), with an apparent binding affinity of 27 nM. Other recombinant NMO antibodies had substantially rapid washout and reduced binding affinity (examples shown in FIG. 7B, right). Slow rAb-53 washout was verified in live cells expressing AQP4. FIG. 7C shows rAb-53 binding over 5-10 minutes, without measurable washout over 3 hours.
- Point mutations in the Fc portion of rAb-53 were introduced in order to inhibit CDC (K322A), ADCC (K326W/E333S) or both (L234A/L235A), while preserving the AQP4- binding Fab sequences (FIG. 8A). Introduction of these mutations did not affect antibody binding to AQP4, with representative surface plasmon resonance data for one of the mutated antibodies shown in FIG. 8B. As expected, the Fc mutations did not significantly alter on or off binding rate constants or reduce binding affinities.
- FIG. 8C shows that a 5-fold excess of each of the mutated antibodies, as well as non-mutated rAb-53, blocked the binding of Cy3 -labeled rAb-53 to AQP4-expressing cells.
- a non-AQP4-specific (isotype control) monoclonal recombinant antibody had no effect.
- human NMO serum which contains a polyclonal mixture of NMO-IgGs, blocked binding of Cy3-labeled rAb-53 (one of five representative human NMO sera shown in FIG. 8D), as did other monoclonal NMO antibodies (rAb-186 shown in FIG. 8D).
- Non- NMO (control) human serum had no effect.
- FIG. 9A shows a live/dead cell assay in which live cells are stained green and dead cells red. Incubation of AQP4-expressing cells with rAb-53 and complement together caused extensive cell killing. The rAb-53 mutants K322A and L234A/L235A, which are deficient in complement Clq activation, caused little cell killing, whereas K326W/E333S, which has intact complement binding, caused cell killing. In control studies, complement or rAb-53 alone did not cause cell killing, nor did rAb-53 and complement together when incubated with AQP4 null cells (not shown). FIG. 9B shows that a five-fold molar excess of K322A or L234A/L235A greatly reduced cell killing by rAb-53 with complement.
- FIG. 9C shows that rAb-53 mutants K322A and L234A/L235A blocked complement-mediated cell killing by NMO sera from different NMO patients (representative data from 3 of 6 patient sera shown). Control (non-NMO) serum did not cause cell killing. Therefore, the aquaporumabs rAb53-K322A and L234A/L235A block binding of different NMO-IgGs and consequent cell killing, probably by steric hindrance at the AQP4 surface.
- FIG. 9D shows marked killing by NK-cells in the presence of rAb-53, with little killing by NK-cells in the presence of control antibody or aquaporumab.
- NMO lesions were created in mouse brain in vivo by intraparenchymal injection of IgG purified from NMO serum, together with human complement. At 24 h after injection, there was marked inflammatory cell infiltration (primarily neutrophils), loss of AQP4 and myelin, and vasculocentric complement activation in the injected hemisphere (FIG. 10A).
- FIG. 10B shows data from five pairs of mice in which NMO-IgG from different seropositive NMO patients was injected with or without aquaporumab. Aquaporumab greatly reduced lesion size.
- NMO-IgG purified IgG from NMO patient serum
- human complement human complement
- FIG. 11B summarizes histological scores of NMO lesion severity. Similar protection by aquaporimub was found for rAb-53, and for NMO-IgG from two other NMO patients.
- the data presented here provide evidence of the utility of aquaporumab blocking antibodies for NMO therapy.
- the engineered high-affinity, non-pathogenic, recombinant monoclonal antibodies blocked cell surface AQP4 binding of polyclonal NMO-IgG in NMO patient sera in cell culture, ex vivo spinal cord and in vivo mouse models of NMO, preventing downstream cytotoxicity and NMO lesions.
- monoclonal antibody therapy has been used for a wide variety of targets and diseases, the idea of a non-pathogenic blocking monoclonal antibody is novel, as is the idea of targeting an autoantibody-antigen interaction for therapy of an autoimmune disease.
- NMO is a unique disease ideal for monoclonal antibody blocker therapy because the single target of pathogenic autoantibodies, AQP4, is a plasma membrane protein having a small extracellular footprint compared to antibody size, and pathology is dependent on antibody effector function.
- Intravenous aquaporumab therapy for NMO is potentially useful during acute disease exacerbations to reduce NMO pathology when the blood-brain barrier at the lesion site is open, and perhaps for maintenance therapy to reduce the frequency and severity of exacerbations.
- Intravitreal administration of aquaporumab may be efficacious in limiting retinal ganglion cell loss following optic neuritis in NMO.
- compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
Abstract
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US10654916B2 (en) | 2020-05-19 |
KR20140059168A (en) | 2014-05-15 |
EP2699256B1 (en) | 2017-09-27 |
JP2014514324A (en) | 2014-06-19 |
US11390667B2 (en) | 2022-07-19 |
AU2012245205B2 (en) | 2017-06-01 |
US20210130444A1 (en) | 2021-05-06 |
CA2833785A1 (en) | 2012-10-26 |
EP2699256A1 (en) | 2014-02-26 |
EP2699256A4 (en) | 2014-11-19 |
JP6152090B2 (en) | 2017-06-21 |
US20140170140A1 (en) | 2014-06-19 |
AU2012245205A1 (en) | 2013-10-31 |
CA2833785C (en) | 2022-06-07 |
CN103702682A (en) | 2014-04-02 |
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